Our long-term goal is to elucidate the endocytic trafficking pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) in order to develop a strategy to correct defective endocytic trafficking of deltaF508-CFTR in individuals with Cystic Fibrosis (CF). DeltaF508, the most common mutation in CF causes: (1) retention of CFTR in the endoplasmic reticulum (ER), (2) reduced open probability of the CFTR CI channel, and (3) decreased plasma membrane half-life of CFTR. Thus, correction of the defects caused by the deltaF508 mutation will require a combination therapy that includes: (1) increased exit from the ER (2) increased CI channel activity, and (3) increased plasma membrane half-life. Of these points, particularly little is known about the mechanisms that regulate the plasma membrane half-life of delta508-CFTR. The hypothesis to be tested in this proposal is that the short plasma membrane half-life of deltaF508-CFTR compared to wt-CFTR results from altered regulation of endocytic trafficking of deltaF508-CFTR. To test this hypothesis we propose three specific aims: Specific Aim #1. Test the hypothesis that the short plasma membrane half-life of deltaF508-CFTR compared to wt-CFTR results from altered endocytic trafficking (increased endocytosis and/or decreased endocytic recycling) of deltaF508-CFTR. The goal of this specific aim is to determine whether the decreased plasma membrane half-life of deltaF508-CFTR is caused by alterations in endocytosis and/or endocytic recycling of CFTR; Specific Aim #2. Test the hypothesis that Rab5a and Rab4 regulate endocytic trafficking of CFTR. The goals of this specific aim are to determine whether these proteins regulate the endocytic trafficking of CFTR and whether altered expression of Rab5a and Rab4 is responsible for the short plasma membrane half-life of deltaF508-CFTR; Specific Aim #3. To characterize the interactions between Rab5a and Rab4 with CFTR. The goals of this specific aim are to determine if Rab5a and/or Rab4 interact directly with CFTR, whether the deltaF508 mutation alters the binding affinities between the Rab5a and Rab4 and CFTR, and to identify the Rab5a and Rab4 interacting proteins that may regulate the endocytic trafficking of CFTR. We anticipate that these studies will elucidate the cellular mechanisms that control the plasma membrane half-life of deltaF508-CFTR. Furthermore, we anticipate that understanding these mechanisms will lead to novel therapeutic strategies for CF and other common diseases that, similar to CF, result from abnormal regulation of protein trafficking.